I need to integrate Gal1 promoter in front of CDC48p. The problem is that i dont know the right carbon source to include on my G418 plate. I have tried varying concentration of Glucose and Galactose on the plate. After transformation I did see some little colonies but none contain my insert. I tried with other different concentration, unfortuantely no colony appear after three days of transformation. Like I said the problem is either using a combination of Galactose and Glucose or Galactose alone and at what concentration should they be in the plate and in the media. Please help me out.
The gene is essential. If you plate on a plate containing glucose, the inhibition of the gal promoter will lead to cell death even if you have galactose and glucose in combination. Using galactose alone will also be problematic since the cells will grow very slowly when using galactose and overexpression of CDC48 in itself will make them grow even slower. You should use a 1%raffinose/2% galactose plate and adjust the galactose concentration as needed.
Thanks Hanna Alalam for your intellectual contribution. I´m waiting for more response before taking the next step.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating